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Production of infectious hepatitis C virus in cell culture has become possible because of the unique properties of the JFH1 isolate. However, virus titers are rather low, limiting the utility of this system. Here we describe the generation of cell culture-adapted JFH1 variants yielding higher titers of infectious particles and enhanced spread of infection in cultured cells. Sequence analysis of adapted genomes revealed a complex pattern of mutations that differed in two independent experiments. Adaptive mutations were observed both in the structural and in the nonstructural regions, with the latter having the highest impact on enhancement of virus titers. The major adaptive mutation was identified in NS5A, and it enhanced titers of three intergenotypic chimeras consisting of the structural region of a genotype 1a, 1b, or 3a isolate and the remainder of the JFH1 isolate. The mutation resides at the P3 position of the NS5A-B cleavage site and slows down processing, implying that subtle differences in replication complex formation appear to determine the efficiency of virus formation. Highly adapted JFH1 viruses carrying six mutations established a robust infection in uPA-transgenic SCID mice xenografted with human hepatocytes. However, the mutation in NS5A which enhanced virus titers in cell culture the most had reverted to wild type in nearly half of the viral genomes isolated from these animals at 15 weeks postinoculation. These results argue for some level of impaired fitness of this mutant in vivo.
The hepatitis C virus (HCV) is an enveloped virus of the genus Hepacivirus within the family Flaviviridae (52). HCV isolates can be classified into six major genotypes that differ in their nucleotide sequence by 30 to 35%, and within these genotypes, several subtypes can be defined (47). Viral infection most often becomes persistent and causes acute and chronic liver disease (22, 46). At present, more than 170 million people suffer from chronic hepatitis C (44). Current treatment consists of a combination of polyethylene glycol-conjugated alpha interferon and ribavirin (44), but success rates are limited and the outcome of therapy is very dependent on the genotype of the infecting virus (13).
The genome of HCV is a linear, single-stranded RNA molecule of positive polarity with a size of ~9.6 kb, and it is flanked at the 5′ and 3′ ends by noncoding regions (NCRs). Both NCRs play an important role in the initiation of RNA synthesis by the viral RNA-dependent RNA polymerase (RdRp) NS5B (16, 18, 27). The RNA genome carries a long open reading frame of about 3,000 amino acids (aa) that is co- and posttranslationally cleaved by cellular and viral proteases into the structural proteins core, E1, and E2, followed by p7 and the nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (3). Expression of the polyprotein is initiated at an internal ribosome entry site, comprising most of the 5′ NCR and the 5′-proximal core coding region (41). The core protein forms the viral nucleocapsid, which is surrounded by the lipid envelope, into which E1/E2 glycoproteins are embedded. Three groups reported that p7 can form oligomers and has ion channel activity (21, 42, 45), suggesting that it belongs to the family of viroporins. More recently, it was shown that p7 is required primarily for efficient assembly and release of infectious virus particles (23, 49). Cleavage between NS2 and NS3 is mediated by the NS2-3 protease, whereas all cleavages C terminal of NS3 are mediated by the N-terminal NS3/4A serine-protease complex. In addition, the C-terminal domain of NS3 functions as a helicase/nucleoside triphosphatase. NS4B induces the formation of membranous vesicles, which seem to contain the viral replication complex (20). NS5A is a phosphoprotein that can be found in two phosphorylated forms (24). Since many cell culture adaptive mutations enhancing replication affect serine residues within NS5A and thereby reduce its hyperphosphorylation, it is assumed that the phosphorylation state of NS5A modulates the efficiency of HCV RNA replication (1, 15, 40). NS5B is the RdRp. The enzyme lacks a proofreading function, which is the reason for the high genetic variability of HCV (reviewed in reference 14).
Studies of the complete HCV replication cycle have become possible with the recent development of a cell culture system that supports production of infectious HCV particles. The system is based on the transfection of the genotype 2a genome JFH1, which replicates extraordinarily efficiently without requiring adaptive mutations (53, 55). However, virus titers released from transfected or infected cells are rather low, reaching at maximum 104 50% tissue culture infectious doses (TCID50) per ml. An improvement of this system has been the generation of virus chimeras consisting essentially of the JFH1 replicase (NS3 to NS5B) fused to the core-to-NS2 region of the HCV J6 isolate (32, 43). Highest virus titers can be achieved with Jc1, which is an intragenotypic J6/JFH1 chimera (43).
In order to improve the JFH1 cell culture system and to gain insight into the determinants of efficient virus production, we adapted authentic wild-type JFH1 (JFH1wt) viruses to the human hepatoma cell line Huh7.5. We identified a major adaptive mutation in NS5A which slows cleavage at the NS5A-B site and enhances virus production of several intragenotypic chimeras. We demonstrate that a highly adapted JFH1 genome is viable in vivo but that some mutations continuously revert to wild type.
All cell lines were grown in Dulbecco's modified minimal essential medium (DMEM) (Life Technologies, Karlsruhe, Germany) supplemented with 2 mM l-glutamine, nonessential amino acids, 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 10% fetal calf serum. The experiments were performed either in Huh7-Lunet cells, supporting high-level RNA replication, or in Huh7.5 cells, which are highly infectible (6, 17).
The NS5B protein was expressed and purified as reported recently (5) to raise rabbit antiserum. The antiserum was further purified via binding to membrane-bound NS5B protein followed by concentration using Centricon Plus-20 centrifugal filter devices (Millipore, Schwalbach, Germany) (for detailed information, see reference 38).
All nucleotide and amino acid numbers refer to the JFH1 genome (GenBank accession no. AB047639). The chimeras Con1/C3, H77/C3, 452/C6, and Jc1 and the JFH1/ΔE1-E2 construct have been described recently (43). Amino acid substitutions were introduced by PCR-based site-directed mutagenesis, and amplified DNA fragments were analyzed by automated nucleotide sequencing by using an ABI 310 sequencer (Applied Biosystems, Darmstadt, Germany). For replication analysis, a point mutation was introduced into pFKI389Luc-EI/NS3-3′/JFH-dg. This plasmid contains the T7 promoter sequence fused to nucleotides 1 to 389 of the JFH1 consensus sequence, followed by the firefly luciferase gene, the encephalomyocarditis virus internal ribosome entry site, the NS3-to-NS5B coding sequence, the 3′ NCR of JFH1, the hepatitis delta virus genomic ribozyme (dg), and the T7 terminator sequence (Volker Lohmann, unpublished).
In vitro transcripts of the individual constructs were generated by linearizing 10 μg of the respective plasmid by digestion for 1 hour with either MluI (full-length and subgenomic JFH1, H77/C3, and Jc1) or AseI (Con1/C3 and 452/C6). Plasmid DNA was extracted with phenol and chloroform and, after precipitation with ethanol, dissolved in RNase-free water. In vitro transcription reaction mixtures contained 80 mM HEPES (pH 7.5), 12 mM MgCl2, 2 mM spermidine, 40 mM dithiothreitol (DTT), a 3.125 mM concentration of each nucleoside triphosphate, 1 U of RNasin (Promega, Mannheim, Germany) per μl, 0.1 μg plasmid DNA/μl, and 0.6 U of T7 RNA polymerase (Promega) per μl. After incubation for 2 h at 37°C, 0.3 U of T7 RNA polymerase/μl reaction mixture was added, followed by another 2 h of incubation at 37°C. Transcription was terminated by addition of 1.2 U of RNase-free DNase (Promega) per μg of plasmid DNA and 30 min of incubation at 37°C. The RNA was extracted with acidic phenol and chloroform, precipitated with isopropanol, and dissolved in RNase-free water. Denaturing agarose gel electrophoresis was used to check RNA integrity, and the concentration was determined by measurement of the optical density at 260 nm.
Single-cell suspensions were prepared by trypsinization, and phosphate-buffered saline (PBS)-washed Huh7-Lunet cells were resuspended at 1 × 107 cells per ml in Cytomix (51) containing 2 mM ATP and 5 mM glutathione, whereas Huh7.5 cells were resuspended at 1.5 × 107 cells per ml. Unless otherwise stated, 5 μg of in vitro-transcribed RNA was mixed with 400 μl cell suspension and electroporated with a Gene Pulser system (Bio-Rad, Munich, Germany) in a cuvette with a gap width of 0.4 cm (Bio-Rad) at 960 μF and 270 V. Cells were immediately transferred to 20 to 25 ml complete DMEM and seeded as required for the assay.
Huh7-Lunet cells were electroporated as described above with 5 μg of JFH1wt RNA transcript, and supernatants of Huh7-Lunet cells harvested 96 h posttransfection were used to infect Huh7.5 cells in a 12-well format. Nearly confluent monolayers were trypsinized and seeded in a six-well format. Further cell passages (cp) were performed in the larger 6-well cell culture format. Overall, 11 cp were performed in the first adaptation experiment (JFH1/adpt1 virus) and 6 in the second adaptation experiment (JFH1/adpt2 virus). In addition to cell passaging, virus-containing supernatants were used to infect new Huh7.5 target cells. Supernatant passages (sp) were repeated in total 13 times for JFH1/adpt1 or 4 times for JFH1/adpt2. During the adaptation process, cell culture supernatants were continuously assessed for infectious virus via indirect immunofluorescence or TCID50 assay, and cells were used for preparation of total RNA (see below).
Total RNA was isolated from a 6-cm-diameter dish of confluent Huh7.5 cells infected with the adapted virus population by using the Nucleo Spin RNAII kit (Macherey-Nagel, Düren, Germany) as recommended by the manufacturer. One microgram total RNA and 50 pmol of primer A9482 (5′-GGA ACA GTT AGC TAT GGA GTG TAC C-3′) were used for cDNA synthesis by using the Expand-RT system (Roche, Mannheim, Germany) as recommended by the manufacturer. Two to four microliters of the reaction mixture was used to amplify the complete open reading frame in two overlapping fragments with the Expand Long Template PCR kit (Roche) according to the instructions of the manufacturer. To amplify the 5′ half of the HCV genome, the PCR was performed with primers S59-EcoRI (5′-TGT CTT CAC GCA GAA AGC GCC TAG-3′) and A4614 (5′-CTG AGC TGG TAT TAT GGA GAC GTC C-3′), and the PCR product was inserted into pFK-I389Luc-EI/NS3-3′/JFH1-dg after restriction with EcoRI and SpeI. The 3′ half of the HCV genome was amplified with primers S3813 (5′-GGA CAA GCG GGG AGC ATT GCT CTC-3′) and A9466-MluI (5′-AGC TAT GGA GTG TAC CTA GTG TGT GCC-3′), and after restriction with SpeI and MluI, the fragment was inserted into pFK-I389neo/NS3-3′/Con1 (34). Sequence analysis was performed with a set of primers covering the complete HCV open reading frame.
Amplification of RNA present in mouse sera was performed by using a slightly modified protocol. Ten microliters total RNA isolated as described above was reverse transcribed using primer A9482, and the first PCR amplification was performed as described above. Because of low RNA levels, nested PCR was required. To amplify the 5′ half of the HCV genome, primers S1298-EcoRI (5′-AAG AAT TCA TTT CGA CCT TGA AGG GGT CCT CG-3′) and A4614 were used. The 3′ half of the HCV genome was amplified with primers S4053 (5′-GGT ACT TGC ATG CTC CAA CTG GCA G-3′) and A9269-MluI (5′-AAA CGC GTT GAG CTT GGT CTT CAC CGC CCA AT-3′). Amplified fragments were inserted into the vectors given above. Alternatively, primers S6689-EcoRI (5′-AAG AAT TCC CTT GCC AAC TAC CTT CTC CAG AG-3′) and A9269-MluI were used for the 3′ half of the HCV genome in just one PCR round. The PCR product was inserted into pFK-I389Luc-EI/NS3-3′/JFH1-dg after restriction with EcoRI and MluI.
Huh7.5 cells were seeded onto glass coverslips in 24-well plates at a density of 4 to 6 × 104 cells per well. Infection was performed 24 h after seeding at a multiplicity of infection (MOI) of 0.3 TCID50/cell, and medium was exchanged at 4 h postinfection. Cells were fixed 24, 48, 72, and 96 h postinfection. For fixation, cells were washed once with PBS and fixed with 500 μl of 4% paraformaldehyde for 10 min at room temperature. Cells were washed three times with PBS, permeabilized by 5 min of incubation in 500 μl of 0.5% Triton X-100 in PBS, and washed three times with PBS prior to blocking with PBS supplemented with 5% normal goat serum for 1 h. Immunostaining of NS3 was performed by using a rabbit polyclonal serum at a dilution of 1:1,000 in PBS with 5% normal goat serum for 45 min. After three washes with PBS, bound primary antibodies were detected by using goat antibodies conjugated to Alexa-Fluor 546 at a dilution of 1:1,000 in PBS containing 5% normal goat serum for 30 min in the dark. DNA was stained with DAPI (4′,6′-diamidino-2-phenylindole dihydrochloride) (Molecular Probes, Karlsruhe, Germany) for 1 min. Finally, cells were washed three times with PBS and once with water and mounted on glass slides with Fluoromount G (Southern Biotechnology Associates, Birmingham, AL).
Quantification of luciferase reporter activity was used to determine transient HCV RNA replication as described previously (30). In brief, transfected Huh7-Lunet cells were resuspended in 22 ml complete DMEM, and 2 ml of the suspension was seeded per well of a six-well plate for harvest at 4, 12, 24, 48, and 72 h after transfection (always in duplicates). For assaying the luciferase activity, cells were washed once with PBS, and 500 μl of lysis buffer (0.1% Triton X-100, 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, and 1 mM DTT, pH 7.8) was added. Cells were frozen immediately, and after thawing, lysates were resuspended by pipetting. For each well, 2 × 100 μl lysate was mixed with 360 μl assay buffer (25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT, 2 mM ATP, and 15 mM K2PO4, pH 7.8) and, after addition of 200 μl of a luciferin solution (200 μM luciferin, 25 mM glycylglycine, pH 8.0), measured for 20 s in a luminometer (Lumat LB9507; Berthold, Freiburg, Germany). The kinetics of replication was determined by normalizing the relative light units at the different time points to the respective 4-h value.
HCV core protein in transfected cells or cell culture supernatants was quantified using the Ortho trak-C enzyme-linked immunosorbent assay (ELISA) kit (Ortho Clinical Diagnostics, Neckargemünd, Germany). Huh7-Lunet cells were electroporated with the respective transcript and resuspended in 22 ml culture medium, and 4-ml aliquots were seeded into five 6-cm-diameter cell culture dishes. Cell lysates and cell culture supernatants were analyzed at 4, 12, 24, 48, and 72 h posttransfection. Cell culture supernatants were filtered through 0.45-μm-pore-size filters and used directly for core ELISA, whereas for determination of intracellular core amounts, cells were lysed by addition of 1 ml PBS containing 1% Triton X-100, 1/10,000 volume aprotinin (1 U/ml), 1/1,000 volume leupeptin (4 mg/ml) and 1/100 volume phenylmethylsulfonyl fluoride (100 mM) and cleared at 18,000 × g for 5 min. Depending on the construct and the time of harvest, samples were diluted 1:10 or higher and processed for ELISA according to the manufacturer's protocol. Colorimetric measurements were performed using a Sunrise colorimeter (Tecan Trading AG, Switzerland). The kinetics of replication was determined by normalizing the intracellular core amounts at the different time points to the respective 4-h value. To determine the efficiency of core protein release, the percentage of extracellular with respect to total core protein (the sum of intra- and extracellular core amounts) was calculated.
Viral RNA was isolated from mouse serum using the Nucleo Spin RNA virus kit (Macherey-Nagel, Düren, Germany) or from virus-infected cells using the Nucleo Spin RNAII kit (Macherey-Nagel, Düren, Germany) as recommended by the manufacturer. Three microliters of the RNA sample was used for quantitative RT-PCR analysis using an ABI PRISM 7000 sequence detector system (Applied Biosystems, Foster City, CA). HCV-specific RT-PCRs were conducted in triplicates with the One Step RT-PCR kit (QIAGEN, Hilden, Germany) using the following JFH1-specific probe (TIB Molbiol, Berlin, Germany) and primers (MWG-Biotech, Martinsried, Germany): A-195, 5′-6-carboxyfluorescein-AAA GGA CCC AGT CTT CCC GGC AAT T-tetrachloro-6-carboxyfluorescein-3′; S-146, 5′-TCT GCG GAA CCG GTG AGT A-3′; and A-219, 5′-GGG CAT AGA GTG GGT TTA TCC A-3′. Reactions were performed in three stages by using the following conditions: stage 1, 60 min at 50°C (reverse transcription); stage 2, 15 min at 95°C (heat inactivation of reverse transcriptase and activation of Taq polymerase); and stage 3, 40 cycles of 15 s at 95°C and 1 min 60°C (amplification). The total volume of the reaction mix was 15 μl, and it contained the following components: 2.66 μM 6-carboxy-X-rhodamine (passive reference), 4 mM MgCl2, 0.66 mM deoxynucleoside triphosphates, 0.266 μM HCV probe, 1 μM of each HCV primer, and 0.6 μl enzyme mix. The amount of HCV RNA was calculated by comparison to serially diluted in vitro transcripts.
A total of 2.5 × 105 Huh7-Lunet/T7 cells (1) were seeded in each well of a six-well cell culture plate in complete DMEM supplemented with G418 (1 mg/ml). About 24 h later, cells were transfected with 2.5 μg pTM-NS3-3′JFH1wt, pTM-NS3-3′JFH1-V2440L, or empty vector (pTM1-2) per well. Transfection was performed by using Lipofectamine LTX (Invitrogen) according to the instructions of the manufacturer. After 4 h, cells were washed once with methionine/cysteine-free medium and left in this medium for starvation for 1 h. For radiolabeling, cells were incubated for 90 min in 1 ml methionine/cysteine-free medium supplemented with 2 mM glutamine, 10 mM HEPES, and 150 μCi/ml of Express protein labeling mix (Perkin-Elmer, Boston, MA). Cells were either lysed directly or washed with PBS and incubated in complete DMEM for 1 or 2 h. Cell lysates were prepared by using NPB (50 mM Tris-Cl [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1/10,000 volume aprotinin [1 U/ml], 1/1,000 volume leupeptin [4 mg/ml], and 1/100 volume phenylmethylsulfonyl fluoride [100 mM]) and cleared by centrifugation at 13,800 × g for 15 min at 4°C. The cleared lysates were used for immunoprecipitation using either the NS5A-specific monoclonal antibody 9E10 (32) or a polyclonal NS5B-specific antibody. Immunocomplexes were dissolved in 70 μl of 2× sample buffer (400 mM Tris [pH 8.8], 10 mM EDTA, 0.2% bromophenol blue, 20% sucrose, 3% SDS, and 2% β-mercaptoethanol), separated in an 8% polyacrylamide-SDS gel, and analyzed by autoradiography. HCV-specific bands were quantified by phosphorimaging using the QuantityOne software (Bio-Rad, Munich, Germany).
Virus titers were determined as described elsewhere with slight modifications (32). Huh7.5 target cells were seeded at a concentration of 1.1 × 104 cells per well of a 96-well plate in a total volume of 200 μl complete DMEM. Twenty-four hours later, serial dilutions of virus-containing supernatant were added, with eight wells per dilution. Two to three days later, cells were washed with PBS, fixed for 20 min with ice-cold methanol at −20°C, washed three times with PBS, and then permeabilized and blocked for 1 h with PBS containing 0.5% saponin (PBS-saponin) plus 1% bovine serum albumin, 0.2% dried skim milk, and 0.02% sodium azide. Endogenous peroxidases were blocked by 5 min of incubation at room temperature with PBS containing 0.3% H2O2 (vol/vol). After three washes with PBS and one with PBS-saponin, NS5A was detected with a 1:400 dilution of hybridoma supernatant 9E10 in PBS-saponin for 1 h at room temperature or overnight at 4°C. Cells were washed again three times with PBS and once with PBS-saponin, and bound 9E10 was detected by incubation with peroxidase-conjugated anti-mouse antibody (Sigma-Aldrich, Steinheim, Germany) diluted 1:200 in PBS-saponin. After 1 h of incubation at room temperature, cells were washed three times with PBS and once with PBS-saponin, and peroxidase activity was detected by using the Vector NovaRED substrate kit (Linaris Biologische Produkte GmbH, Wertheim, Germany). Virus titers (TCID50/ml) were calculated based on the method of Spearman and Kärber (25, 48). Infectivity in all cell lysates was measured in the same way by using lysates obtained by three cycles of freezing and thawing (19). Cells were washed once with PBS, scraped into PBS, and collected by centrifugation at 400 × g for 5 min. The cell pellet was resuspended in 500 μl complete DMEM, and cells were lysed by three rapid freeze-thaw cycles by using liquid nitrogen and a 37°C thermoblock, respectively. Cell debris was pelleted by centrifugation for 10 min at 10,000 × g, and the supernatant was used for titration as described above.
The mouse study was conducted at Ghent University Hospital, with protocols approved by the Ethical Committee and Animal Ethics Committee of the Ghent University Faculty of Medicine. Transgenic SCID mice that overexpress the uPA gene under the control of an albumin promoter were used for xenotransplantation with primary human hepatocytes as described elsewhere (35, 36, 37). Mice were inoculated by intraperitoneal injection of 2.8 × 105 TCID50 of a straight culture supernatant stock of JFH1mut1-6 virus. The virus stock was generated by electroporation of 5 μg RNA into Huh7-Lunet cells, and virus titers in the supernatant were determined as 7.6 × 105 TCID50/ml.
To adapt JFH1wt virus to cell culture, we infected Huh7.5 cells with the plain supernatant of Huh7-Lunet cells transfected with the JFH1 RNA genome (as described in Materials and Methods) and passaged the cells up to 11 times (Fig. (Fig.1).1). Huh7.5 cells were used because they support high levels of HCV RNA replication, which is assumed to be due to a defect in the induction of the double-stranded RNA response (6, 50). The second attribute of Huh7.5 cells is high-level CD81 expression on the cell surface, which is a critical prerequisite for efficient infection and thus for cell culture adaptation (discussed in references 4 and 29). After 2 cp, growth of the cells had already become arrested (Fig. (Fig.1),1), likely due to cytopathogenicity of JFH1, which at this time replicated in most cells in the culture dish (data not shown). However, virus titers released from passaged cells did not change up to the cp 8. After 8 cp, virus-containing supernatant was passaged multiple times on naïve Huh7.5 cells, resulting in a virus pool in which titers had been increased about 1,000-fold compared to the parental JFH1 strain (Fig. (Fig.11).
To further characterize the adapted virus population obtained in this first adaptation experiment (JFH1/adpt1 virus), Huh7.5 cells were infected in parallel with JFH1/adpt1 or with JFH1wt at an MOI of 0.3 TCID50/cell, and infection and virus spread were determined by indirect immunofluorescence. As illustrated in Fig. Fig.2A,2A, JFH1/adpt1 spreads much faster in cell culture than JFH1wt. At 96 hours postinfection, nearly 100% of the JFH1/adpt1-infected cells but only a few JFH1wt-infected cells were NS3 positive. Likewise, cells infected with the adapted viruses released about 106 TCID50/ml at 4 days postinfection (Fig. (Fig.2B),2B), whereas JFH1wt titers were undetectable with this assay, most likely due to the very low MOI used for the initial inoculation. Infections with a higher MOI were not possible due to the very low virus titers obtained with JFH1wt. However, when using the more sensitive quantitative RT-PCR assay, viral RNA was detectable in JFH1wt-inoculated cells (Fig. (Fig.2C).2C). Nevertheless, viral RNA levels were almost 1,000-fold higher in case of JFH1/adpt1-inoculated cells.
To identify the mutations responsible for the enhanced virus production, HCV genomes isolated from infected cells were reverse transcribed and amplified in two fragments, and amplicons were sequenced as explained in Materials and Methods. From each amplicon, at least three clones were analyzed (Table (Table1).1). Only those mutations that were detected at least in two different cDNA clones and that led to an amino acid substitution were considered. Initially, the virus genomes obtained after 8 cp and 8 sp were sequenced completely, and mutations were found both in the structural region (core, E1, and E2) and in the NS region (NS5A and NS5B). In analysis of samples taken after cp 3 and 6, we found that only mutation 2 in E1 and mutation 5 in NS5A were fixed (Table (Table1).1). All other mutations found in JFH1/adpt1 emerged between cp 6 and the end of the experiment (8 cp plus 8 sp) (Table (Table11).
To determine which of these mutations enhanced virus production, we inserted each mutation individually and in several combinations into the JFH1wt genome (Fig. (Fig.3A).3A). The impact of these mutations on RNA replication was determined by electroporation of Huh7-Lunet cells with the respective in vitro transcript and measurement of core protein accumulation in transfected cells during a 3-day period (Fig. (Fig.3B).3B). Core protein amounts determined 4 h after transfection were used to correct for transfection efficiency, because at this time point replication is not yet measurable and core protein thus is generated from input RNA. As shown in Fig. Fig.3B,3B, all constructs replicated to very similar levels, showing that the mutations do not affect RNA replication.
Next, we verified whether the engineered mutations affect virus production from transfected Huh7-Lunet cells by determining the percentage of extracellular with respect to total core protein (Fig. (Fig.3C).3C). Interestingly, V2440L, close to the C terminus of NS5A (mutation 5), alone or in combination with the remaining mutations increased core release about 10-fold compared to wild type, whereas the mutations in the structural region had no significant impact. This efficiency of core release is comparable to that obtained with the chimeric genome Jc1 (43), which is the most efficient HCV chimera available thus far. Analogous results were obtained when infectivity titers in cell culture supernatants were determined (Fig. (Fig.3D).3D). Also in this assay, V2440L in NS5A, either alone or in combination with the other mutations, enhanced the overall amounts of released infectivity and accelerated the kinetics of virus release. Already at 12 h posttransfection, infectious virus was detected in culture supernatant, and infectivity titers were about 100-fold higher at 24 h posttransfection than those for the wild type. Interestingly, the mutation in E1 already detected in very early cell passages (mutation 2 in Table Table1)1) had no or only a minor impact on release of core and infectious virus (Fig. 3C and D). The mutation in core had no significant effect on virus titers either alone or together with the other six mutations (data not shown). It should be noted that the Huh7-Lunet cells used for these experiments express only low CD81 levels on the cell surface and thus support virus spread only poorly. Thus, the virus titers measured here are due to release from transfected (primary) cells rather than to release from cells infected by spreading (secondary) virus. In summary, mutations in the replicase complex, in particular in NS5A, can have profound effects on virus production without affecting RNA replication. This result argues for a cross talk between the structural proteins and the replicase complex to achieve efficient virus assembly.
Having identified NS5A as a critical element for efficient virus production, we wanted to know whether NS5A is the only determinant of or whether mutations in other HCV genes may also enhance virus assembly. We therefore performed a second adaptation experiment with JFH1 and Huh7.5 cells and passaged infected cells six times, followed by 4 sp (Table (Table1).1). As in the first experiment, passaged JFH1-containing cells produced much higher virus titers, and virus spread in the culture was much faster than the spread observed with JFH1wt (data not shown). The complete open reading frames of the adapted virus pool were amplified in two fragments, and three clones of each amplicon were subjected to nucleotide sequence analysis. Several conserved amino acid substitutions were found, which are summarized in Table Table11 (adpt2). To our surprise, none of the mutations identified in the first adaptation experiment reappeared in the second one. In addition, most mutations in JFH1/adpt2 resided in other proteins, namely, p7 and NS3. Only one mutation resided in NS5A.
To define those mutations contributing to adaptation, we inserted each mutation individually or all three together into the JFH1wt genome (Fig. (Fig.4A)4A) and characterized the mutants for RNA replication, core release, and production of infectious virus. None of the mutations had a measurable effect on RNA replication as indicated by the equivalent levels of core protein accumulation in transfected cells (Fig. (Fig.4B).4B). The most potent effect on the release of core (Fig. (Fig.4C)4C) and infectivity (Fig. (Fig.4D)4D) was found with the mutation in p7 (mut7). Comparable to the V2440L mutation in NS5A, the overall titer was increased up to 100-fold, especially at early time points. Likewise, the kinetics of virus release was accelerated, and infectious virus particles were already detected at 12 h postelectroporation (Fig. (Fig.4D).4D). In contrast the mutation in the NS3 helicase (mut8) had only a minor effect on particle production, whereas the mutation in NS5A (mut9) had no or a slightly negative effect. The combination of all three mutations (mut7-9) led to only slightly higher virus production than with the genome carrying only the p7 mutation. In line with very recent studies (23, 49), these results identify p7 as another important determinant for efficient virus production, and they suggest that numerous factors, such as p7 or NS5A, are crucial for virus assembly and release.
Attempts to increase virus titers further were of limited success. When we combined the most efficient adaptive mutations of the first and second adaptation experiments (V2440L and N765D or V2440L, N765D, and I1316V), the mutations acted only additively and enhanced virus titers to the level of JFH1mut4-6 (data not shown).
We and others have recently described the construction of virus chimeras (32, 43) consisting of the region encoding core to the N- or C-terminal region of NS2 (designated positions C3 and C6, respectively) of the isolates Con1 (gt 1b), H77 (gt 1a), 452 (gt 3a), and J6 (gt 2a) fused to the remainder of the JFH1 isolate (Fig. (Fig.5A).5A). However, apart from the very efficient intragenotypic J6/JFH1 chimera (designated Jc1), virus titers produced by the intergenotypic chimeras are rather low (43). Since all these chimeras are based on the JFH1 replicase region and thus contain the NS5A of JFH1, we investigated whether the main adaptive mutation V2440L also increases virus titers of these chimeras. Huh7-Lunet cells were transfected with in vitro transcripts of the mutated chimeras depicted in Fig. Fig.5A,5A, and RNA replication was determined by analyzing the accumulation of intracellular core protein. As shown in Fig. Fig.5B,5B, for each pair of constructs, the wild type and the corresponding mutant replicated to comparable levels. However, the NS5A mutation increased both the efficiency of core protein release (Fig. (Fig.5C)5C) and the release of infectivity (Fig. (Fig.5D)5D) of all three intergenotypic chimeras. The most prominent enhancement could be achieved with the gt 3a/JFH1 chimera (452/C6), where infectivity titers were increased more than 100-fold. In contrast, titers of the already very efficient intragenotypic Jc1 chimera could not be increased further. This may be due to a limitation of the host cell or to a limitation of the virus genome. Arguing against the latter assumption, insertion of the V2440L substitution into the Jc1 genome accelerated kinetics of virus release so that infectious particles could be detected already at 6 h posttransfection instead of 8 h as is the case for Jc1 without the mutation (data not shown).
Having identified V2440L in NS5A as a major adaptive mutation, we wanted to know by which mechanism this mutation enhances virus production. In the first set of experiments, we wanted to exclude subtle differences in RNA replication associated with the V2440L mutation, which might not be visible by determining intracellular core protein accumulation. We therefore generated bicistronic subgenomic JFH1 replicons carrying a luciferase reporter gene in the first cistron and the wild-type replicase (NS3-NS5B) or the replicase carrying the V2440L substitution in NS5A. However, with this highly sensitive and quantitative method we did not detect differences in RNA replication between the wild type and the mutant, with respect to either replication kinetic or overall replication levels (Fig. (Fig.6A6A).
Next, we assessed whether the V2440L substitution has an effect on virus release. Huh7-Lunet cells were transfected with JFH1wt or JFH1mut5 RNA transcripts, and titers of intra- and extracellular virus were determined at 48 h postelectroporation (Fig. (Fig.6B).6B). In line with our previous result (Fig. (Fig.3D),3D), virus titers of JFH1mut5 were approximately 10-fold higher than titers of JFH1wt. In addition, the titers of intracellular virus were increased approximately 10-fold in case of the mutant. We conclude that the mutation in NS5A enhances primarily virus assembly rather than release of infectious virus particles.
The V2440L mutation is located at the P3 position of the cleavage site between NS5A and NS5B and thus may affect polyprotein processing. We therefore compared the cleavage efficiencies at the NS5A-B site for JFH1mut5 and JFH1wt by using pulse-chase experiments. To achieve expression levels that are sufficient for this analysis, we used a T7-based expression system in which T7 promoter-driven NS3-to-NS5B constructs were transfected into Huh7-Lunet/T7 cells. Proteins were radiolabeled for 90 min with [35S]methionine/cysteine and harvested either immediately or after a 1- or 2-h chase period. Immunoprecipitations were conducted with NS5A- or NS5B-specific antibodies (Fig. 6C and D, respectively) and immunocomplexes were analyzed on an 8% sodium dodecyl sulfate-polyacrylamide gel. Mature NS5A and NS5B could be detected for the wild type and the mutant. In addition, uncleaved NS5AB with a size of approximately 130 kDa and a precursor of approximately 170 kDa (presumably NS4B5AB) could be detected. Only small amounts of these precursors were found in the case of JFH1wt, reflecting rapid cleavage at the NS5A-B site as described for genotype 1 polyprotein processing (2, 31). In the case of the mutant, the amounts of these precursors were significantly increased concomitant with reduced amounts of mature NS5A and NS5B (quantifications of protein levels are given at the bottom of Fig. 6C and D). These results argue for a delayed cleavage kinetics at the NS5A-B site due to the V2440L substitution at the P3 position. However, the impaired cleavage did not lead to an altered NS5A phosphorylation pattern, as deduced from the comparable ratio between basal and hyperphosphorylated NS5A (lower and upper bands in Fig. Fig.6C,6C, respectively).
We have recently shown that Con1 genomes carrying replication-enhancing mutations are attenuated in vivo (8). We therefore wanted to know whether the highly cell culture-adapted JFH1mut1-6 virus is viable in vivo. To address this question, we took advantage of the uPA-SCID mouse model, in which immunodeficient transgenic mice are xenografted with primary human hepatocytes, which are then permissive for HCV infection (35). Two mice (animals K371− and K371L) were each inoculated with 2.8 × 105 TCID50 of JFH1mut1-6 virus. At 2 weeks after infection, the sera of both animals contained high viral loads of HCV RNA (about 107 copies/ml) (Fig. (Fig.7A).7A). Viral loads remained above 5 × 105 HCV RNA copies/ml until week 15 postinfection, when animals were sacrificed. This result suggested that the highly adapted JFH1 genome is viable in vivo. However, it was also possible that at least some of the mutations had reverted to wild type. We therefore analyzed whether the introduced six adaptive mutations of JFH1mut1-6 were retained in the viral genomes. Virus RNA was isolated from sera of the sacrificed animals, and HCV cDNA was amplified and cloned in two fragments as described in Materials and Methods. The first fragment spanned the coding region of E1 to NS3, and the second fragment spanned the region encoding most of NS5A to NS5B. At least four cDNA clones of each amplicon and each animal were analyzed (Fig. (Fig.7B).7B). In all four analyzed E1/NS3 clones of animal K371−, mutations 1, 2, and 3 were conserved. In contrast, in animal K371L, in all sequenced clones, mutation 2 in E1 had reverted to wild type, whereas mutations 1 and 3 were retained. A different picture was observed with the NS5A-5B amplicons. Two of seven analyzed clones recovered from animal K371− contained the two mutations in NS5A and the one in NS5B. However, in the five other clones the first mutation in NS5A was retained, the major adaptive mutation (V2440L) was reverted to wild type, and the methionine substitution in NS5B was replaced by threonine. In animal K371L this combination was found once. In six other sequences, the two NS5A mutations were retained, and the threonine substitution in NS5B and in addition a new mutation in NS5A (S2390P) appeared. In summary, in nearly half of the sequenced virus populations isolated from both animals, the major adaptive mutation in NS5A was reverted to wild type. This finding suggests that the highly adapted JFH1 genome is viable in vivo but has an impaired fitness.
In this study we successfully adapted infectious HCV particles of the genotype 2a JFH1 strain to Huh7.5 cells. Adapted virus genomes retained wild-type RNA replication levels (Fig. (Fig.3B3B and and4B)4B) but produced 10- to 100-fold higher virus titers than the wild-type virus (Fig. (Fig.3D3D and and4D).4D). One major adaptive mutation (V2440L) resided at the P3 position of the NS5A-B cleavage site and reduced cleavage kinetics. This observation is in line with findings of Kim and coworkers (26), who described that the NS3/4A protease of a genotype 1b isolate prefers valine over leucine at the P3 position. Furthermore, in none of the HCV genome sequences deposited in the European HCV database (euHCVdb) (12) was a P3 leucine found. Apart from valine, which is the most common P3 residue, isoleucine or alanine residues are found with some isolates. Nevertheless, enhancement of virus production by reduced processing at the NS5A-B site appears to be a more general mechanism, because insertion of V2440L into three different HCV chimeras resulted in increased virus titers. Moreover, in an independent adaptation experiment with the Con1/C3 chimera, the V2440L substitution was also found (A. Kaul, I. Woerz, and R. Bartenschlager, unpublished).
Thus far, we can only speculate on how reduced NS5A-B cleavage increases virus assembly. Altered processing kinetics may affect folding and maturation of NS5A or the replicase complex. This may affect interaction of the replicase with the structural proteins, in particular core protein, during virus assembly. In this context we note that subtle differences in polyprotein processing were shown to affect the phosphorylation status of NS5A, arguing for a tight regulation of polyprotein processing and “maturation” of the cleavage products forming the replicase (28, 39), which in turn may affect the cross talk between the replicase and the assembly machinery.
In the first adaptation experiment, in addition to the V2440L substitution, five further mutations were detected. Three of them resided in the structural region (core, N16D; E1, I372V and I374T; and E2, I422L) and two in the NS region (NS5A, V2153A; and NS5B, V2941M). While each of the last two mutations slightly enhanced virus titers (data not shown), the mutations in the structural region had no impact on virus production when tested alone (data not shown). We observed only a slight enhancement of virus production when the I374T substitution in E1 was combined with the major adaptive mutation V2440L (mut2+5 in Fig. Fig.3D).3D). This could be traced back to the adaptation history, because both mutations coexisted in genomes isolated after the third cell passage (Table (Table1).1). The I374T substitution is localized in the transmembrane domain of E1, which also is required for proper formation of E1-E2 heterodimers. This mutation could therefore play a role in the biogenesis of entry-competent E1-E2 complexes (11). The only mutation found in NS5B (V2941M, corresponding to aa 499 of NS5B) resides in the N-terminal part of the α-helix thumb. The mutation could affect the allosteric GTP binding site (7), but given its location on the surface of the molecule, the methionine substitution may also affect interaction with some viral or cellular factor. This assumption is affirmed by findings of Cai and coworkers. Their mutational analysis of aa 499 and other amino acids involved in GTP binding demonstrated that mutations of the GTP binding site that do not affect in vitro RdRp activity still can impair or even ablate HCV RNA replication in cell culture (9).
In the second adaptation experiment, we identified three other mutations that also enhance the release of infectious virus (Fig. (Fig.4D)4D) without affecting RNA replication (Fig. (Fig.4B).4B). This result suggests that virus assembly is a complex process requiring numerous interactions that may be modified individually to enhance virus formation. A major adaptive mutation was identified in the first transmembrane domain of p7 (N765D). A search in the euHCVdb revealed that this residue appears to be isolate specific. While genotype 1a and 1b isolates have a glycine residue at this position, genotype 2 isolates have a serine residue, except for JFH1. Residue 765 resides in the N-terminal transmembrane α-helix of p7 and may be involved in oligomerization. According to the helical wheel diagram of that part of the protein, one can assume potential hydrophobic binding sites on the face of each helix (10). Thus, position 765 could be involved in a hydrophobic interaction between helices 1 and 2 of adjacent p7 monomers. The major effect of this mutation was acceleration of virus release, arguing that p7 plays a very important role in virus assembly and/or release. In support of this conclusion, we and others recently found that p7 is essential for efficient assembly and release of infectious virions (23, 49). Whether p7 enhances the specific infectivity of virus particles is controversial (49, 54). At least when calculating the ratio of TCID50/ml to extracellular core amounts, which corresponds to the specific infectivity of a virus, the N765D mutant is similar to the wild-type virus.
As we found completely different adaptive mutations in independent experiments, it is not surprising that Zhong and coworkers, who also adapted the JFH1wt isolate to a Huh7.5 subclone, found yet other mutations enhancing virus production and specific infectivity (56). These are located in core, E2, NS3, and NS5A, and among them the G451R mutation in E2 increases infectivity titers 10-fold, whereas the other mutations have no detectable effect. In a recent study, Yi and coworkers adapted different H77/JFH1 chimeras with various crossover points between NS2 and NS3 to Huh7 cells and observed the reappearance of adaptive mutations in several independent experiments (54). Two alternative mutations in p7 and NS3 were primarily detected, which may compensate for incompatibilities between the structural and the NS proteins that were derived from the genotype 1a H77 isolate and the JFH1 replicase. When we adapted several intergenotypic chimeras to cell culture (H77/C3, Con1/C3, and 452/C3), we also repeatedly observed, among others, chimera-specific mutations in p7 and NS3 (A. Kaul, I. Woerz, and R. Bartenschlager, unpublished). The reappearance of some mutations in independent adaptation experiments argues for limited possibilities to compensate for incompatibilities.
When we infected chimeric uPA-SCID mice with JFH1mut1-6 virus, we observed persistent replication for 15 weeks until the experiment was stopped (Fig. (Fig.7A).7A). Two weeks after infection, sera of both animals contained high viral loads (about 107 HCV RNA copies/ml), which remained above 5 × 105 HCV RNA copies/ml until the animals were sacrificed. These findings are in line with results of Lindenbach and colleagues, who observed persistent replication of a J6/JFH1 chimera in uPA-SCID mice (33). As it was recently shown that Con1 genomes carrying replication-enhancing mutations are attenuated in chimpanzees and rapidly revert to wild type (8), in the present study we determined whether the six introduced mutations of JFH1mut1-6 were retained in the HCV genomes circulating in mice 15 weeks after infection. In contrast to the earlier study where the combination of three highly cell culture-adaptive mutations in the Con1 genome completely abolished infectivity in vivo and a genome containing only one adaptive mutation in NS5A was attenuated and rapidly reverted to wild type, in the present study we recovered mainly HCV genomes that still contained some or all of the introduced mutations (Fig. (Fig.7B).7B). While this result suggests that the highly adapted JFH1 genome is viable in vivo, it is remarkable that only about half of the analyzed HCV genomes contained the major adaptive V2440L mutation in NS5A. Interestingly, in HCV genomes of animal K371L, a new mutation arose in NS5A (S2390P) that coexisted with L2440, arguing that the additional mutation may compensate for some attenuating effect of V2440L. In addition, the introduced mutation V2941M in NS5B did not revert to wild type, but in about 85% of the sequenced clones methionine was replaced by threonine. A BLAST search in the euHCVdb database revealed that most isolates possess an alanine residue at position 2941, a valine or a threonine residue is less frequently found, and none of the listed isolates posses a methionine residue at this position. Moreover, in addition to these mutations, a complex and mouse-specific pattern of nonconserved mutations was found, especially in the structural region, which will be investigated in further studies.
In summary, in this study we identified mutations in the NS region which enhance virus production and thus increase virus titers by several orders of magnitude. Our data suggest that the replicase machinery, in particular NS5A, is an important assembly determinant and that subtle alterations of polyprotein processing can have profound effects on virus production. These data will inform future studies on the molecular mechanisms underlying HCV morphogenesis.
We are grateful to Ulrike Herian, Stephanie Kallis, Jennifer Schmitt, Anna-Lena Gamer, and Lieven Verhoye for excellent technical assistance. We thank Nicole Appel, Rahel Klein, and Volker Lohmann for generation and purification of polyclonal NS5B antisera. We thank Charles M. Rice for providing Huh7.5 cells and Timothy L. Tellinghuisen and Charles M. Rice for the NS5A monoclonal antibody 9E10. We thank Thomas Longerich and Peter Schirmacher for help in the analysis of liver histology of infected mice. We are also grateful to Tim Hart and Eike Steinmann for critical reading of the manuscript.
This work was supported by the research program RNS/RNAi of the Landesstiftung Baden-Württemberg (P-LS-RNS30) and a grant from the VIRGIL European Network of Excellence (LSHM-CT-2004-503359). Additional support was provided by a Concerted Action Grant from Ghent University (01G00507).
Published ahead of print on 19 September 2007.